Studies On The Correlation Between Nemo-like Kinase And Breast Carcinoma

Objective1. To investigate the expression of NLK and c-Myb in the breast carcinoma, and analyze the correlation of NLK abnormal expression and the occurrence and development of breast carcinoma.2. To investigate the expression variation of NLK during the proliferation and apoptosis process of breast carcinoma, and investigate the role of NLK during the process. To futher reveal this process appears to involve c-Myb and might provide a novel target for therapeutic intervention in patients with breast cancer.Methods:1. The expression of NLK and c-Myb in8cases of fresh frozen specimens from breast carcinoma and the adjacent breast tissue were detected by Western blot. Immunohistochemical to detect the expression of NLK, c-Myb and Ki67(the proliferation index) in62samples of breast carcinoma tissue and adjacent non-tumor tissue. To analyze the connection between NLK and clinicopathologic datas of breast cacinoma patients, such as age, gender, histological grade, metastasis, ER, PR, Her2, proliferation index and so on.62cases of breast patients were followed up.2. The expression of NLK and c-Myb were detected in normal human breast epithelial cell line HBL-100and two human breast cancer cell lines:MDA-MB-231and MCF-7. MCF-7cells were treated with serum starvation and release for synchronization purpose, the distribution of MCF-7cell cycle was detected by flow cytometer, then detect the expression variation of NLK and c-Myb during the proliferation of MCF-7cell by Western blot. MCF-7cells were transfected with pcDNA3.1-EGFP-NLK. Western blot was used to detect the expression of PCNA (a marker of proliferation) and active caspase-3(a marker of apoptosis). MTT assay determine the biologic effects of the over-expression of NLK expression, the growth rate of the MCF-7cells. Flow cytometry detected the proliferation and apoptosis of MCF-7cells. Western blot was used to detect c-Myb degradation MCF-7cells transfected with pcDNA3.1-EGFP-NLK and expression levels of Bcl-2and c-myc.Results1. Immunohistochemistry and western blot analysis showed that NLK expression in breast carcinoma were lower than that in non-tumor breast tissue. NLK expression was correlated with histological grade(P<0.001), Her2(P<0.029) and Ki67(P<0.001) expression. Correlation analysis showed NLK expression was negatively associated with Ki67(r2=-0.743;p<0.001).2. Western blot was used to detect the expression of NLK and c-Myb in HBL-100, MDA-MB-231and MCF-7cells. MCF-7cell lines displayed lowest abundance of NLK and highest expression level c-Myb among these cell lines. The cell cycle of MCF-7cells was blocked by serum starvation. The expression of NLK was increased. The albumen amount of c-Myb was down-regulated. The MCF-7cells were reentering the cell cycle after serum release and the reverse changes of these proteins were happened. These results analyzed kinetics of NLK and c-Myb expression during cell-cycle progression. We found that NLK inhibits proliferation and promotes apoptosis in MCF-7cells after the cells were transfected with pcDNA3.1-EGFP-NLK. These results thus suggested that NLK was able to change the expression level of c-Myb in MCF-7cell so as to the target gene of c-Myb. We verified that NLK played a role in proliferation and apoptosis of breast cancer by the regulation of c-Myb.Conclusions1. The expression of NLK was downregulated significantly in breast carcinoma. NLK served as an independent prognostic marker for breast cancer. These datas revealed that NLK may play a role in the oncogenesis and development of breast carcinoma.2. During the proliferation and apoptosis process of MCF-7cells, the regulation of c-Myb by NLK played a role in it.